1. Field of the Invention
This invention generally relates to novel particles which are particularly useful for research, diagnostic and therapeutic applications for human and animal health care in the field of cell selection. More specifically the invention relates to novel ferromagnetic dense particles (FDP), and methods of preparation and use thereof, in applications that include selection of cells, cell components, bacteria, viruses, toxins, nucleic acids, hormones, proteins, receptor-ligand complexes, other complex molecules or any combination thereof. The particles can in addition have application in molecular biology and protein purification protocols
2. Background Art
The enhancement, via positive selection or depletion, of a population or subpopulation of cells or cellular-like material in fluid from a human or animal subject can be utilized for a number of applications. For instance in the area of cell therapy, the depletion of tumor cells prior to an autologous stem cell transplant or the depletion of certain immune cells prior to an allogeneic stem cell transplant can prove beneficial to the patient. In diagnostics, the enrichment of rare cells may be useful for early diagnosis of metastatic disease (see for example U.S. Pat. No. 6,365,362; April 2002; Terstappen, L. et al.). In the research arena, both positive and negative selection procedures are powerful tools for cell-based studies and, in the molecular biology arena, selection tools that permit the detection of specific genes or mRNA's are extremely important for both gene discovery and as research tools. Conventional cell enrichment/depletion and other selection technologies are based on either gradient centrifugation procedures well known in the art or solid-phase microparticles (herein used interchangeably with particles) that are linked to binding agents, which separate cells via magnetic attraction or gravity. Microparticles can range in diameter from about 50 nm to about 50 microns and can have either smooth or raged surfaces. Magnetic selection technologies include colloidal superparamagnetic particles (see, for example, those provided by Immunicon Corporation or Miltenyi Biotech) and micron sized particles (such as those provided by Dynal Corporation). Gravity selection technology can include micron size dense particles (HDM), such as those provided by Eligix, a subsidiary of Biotransplant, Inc.
Magnetic Selection:
As is well known in the art, several types of magnetic particles can be prepared, including but not limited to, superparamagnetic and ferromagnetic. U.S. Pat. Nos. 5,411,863 and 5,466,574 (May 1995; Miltenyi, S. and November 1995; Liberti, P. et al., respectively) teach that superparamagnetic particles are the particle of choice for biological selection applications. Superparamagnetic materials have in recent years become the backbone of magnetic selection technology in a variety of health care and bio-processing applications. Superparamagnetic materials are highly magnetically susceptible—i.e. they become strongly magnetic when placed in a magnetic field but rapidly lose their magnetism when the magnetic field is removed. This property makes it easy to isolate cell populations and to resuspend the cells when the magnetic field is removed. Superparamagnetism occurs in ferromagnetic materials when the crystal diameter is decreased to less than a critical value. Such materials, regardless of their diameter (about 25 nm to about 100 microns) have the property that they are only magnetic when placed in a magnetic field. The basis for superparamagnetic behavior is that such materials contain magnetic material in size units below about 20 to 25 nm, which is estimated to be below the size of a magnetic domain. A magnetic domain is the smallest volume for a permanent magnetic dipole to exist. Ferromagnetic materials, on the other hand, are strongly susceptible to magnetic fields and are capable of retaining magnetic properties when the field is removed. Ferromagnetism occurs only when unpaired electrons in the material are contained in a crystalline lattice thus permitting coupling of the unpaired electrons. The prior art teaches that ferromagnetic particles with permanent magnetization have considerable disadvantages (see for example U.S. Pat. No. 5,411,863; May 1995; Miltenyi, S. and U.S. Pat. No. 5,466,574; November 1995; Liberti, P. et al.) for applications in biological material selection, since suspensions of these particles easily aggregate following exposure to a magnetic field due to their high magnetic attraction for each other. For this reason ferromagnetic particles have not been used in the art for biological (cell selection/nucleic acid) applications.
Gravity Selection:
Solid phase microparticles that separate targeted from non-targeted populations on the basis of gravity rather than magnetics have been described (for example, U.S. Pat. No. 5,576,185; November 1996; Coulter, W. et al. and Zwerner, et al. J. Imm. Methods (1997) 14:31-34). Currently, particles that are separated based on gravity are relatively dense and large with diameters between the range of approximately 3 to 10 microns. A known feature of these particles is that because of the density difference between particles and cells, end-over-end mixing allows the particles to pass through a substantial portion of the fluid sample. The particles traverse past the cells of interest and in doing so bind to the targeted cell population without non-specifically binding to non-target cells. This leads to an efficient separation and a high recovery of non-targeted cells. The separation and recovery of non-targeted cells is superior to that found with superparamagnetic selection alone. These dense particles are designed to settle by gravity both as a mixing manner (discussed above) and as a manner to separate the desired population of cells from the remainder of the cell suspension. In fact, previous descriptions (for example U.S. Pat. No. 5,576,185; November 1996; Coulter, W. et al.) teach away from the use of smaller particles in gravity selection. For example, the disclosure of this patent teaches that superparamagnetic particles are intended to be maintained in suspension in the sample and consequently are designed for very slow or substantial elimination of gravity settling in the sample suspension. Typically, well-coated materials below 150 nm will show no evidence of settling for as long as 6 months and even longer (see U.S. Pat. No. 5,622,831; April 1997; Liberti, P. et al.). Thus, superparamagnetic particles are not applicable in gravity selection technology or density difference mixing. Both procedures function optimally at a density difference of at least 2-3 fold between the particles and the target biomaterial when capturing cells and settling by gravity.
Gravity separation addresses several drawbacks inherent in magnetic separation procedures that utilize superparamagnetic particles including non-specific cell loss due to trapping, time of magnetic collection when using colloidal particles, and/or the high magnetic gradients required for collection of colloidal particles. When using superparamagnetic colloidal particles, there often is not sufficient magnetic force to hold the targeted cells at the wall of the chamber. This leads to contamination of the non-targeted cells with targeted cells. Also, sample preprocessing is often required (i.e. removal of red blood cells via density gradients before addition of superparamagnetic particles). However, by using gravity selection, sample preprocessing can often be eliminated and minimal cell loss of non-target cells occurs, but a drawback of the conventional art has been that due to the size of the particles and the nature of gravity mixing, cells with low antigen density are not easily isolated, thus limiting its utility.
Accordingly, it would be desirable to have an effective method of depleting or selecting one or more populations of cells or cell-like material without affecting the remaining populations in the sample, over a broad range of antigen density on the cell surface. The method ideally should be inexpensive, fast, result in high yield and not restricted by the sample volume.